histone H3-H4 and free histone H2A-H2B dimers (Fig. 1D and fig. S2, A to C). The efficiency
of H3-H4 binding increased for ssDNA-bound
RPA, indicating that the RPA–H3-H4 interaction
likely occurs on chromatin (Fig. 1E). In parallel
assays for Asf1, ssDNA did not affect the efficiency of Asf1–H3-H4 binding (Fig. 1E). Domain
mapping studies revealed that Rfa1, and specifically its oligonucleotide/oligosaccharide-binding
fold domain at the N terminus (OB-N), was likely
the major contributing site of the RPA-histone
interaction (fig. S2, D to H).

We used electrophoretic mobility shift assays
(EMSAs) to examine the importance of the RPA–
H3-H4 interaction in the formation of DNA-histone
complexes. To mimic freshly unwound ssDNA at
the replication fork, we designed a fluorescently
labeled substrate that consisted of a 149-base